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Wheat damage by Aelia spp. and Erygaster spp.: effects on gluten and water-soluble compounds released by gluten hydrolysis
Journal of Cereal Science 39 (2004) 187–193 www.elsevier.com/locate/jnlabr/yjcrs
Wheat damage by Aelia spp. and Erygaster spp.: effects on gluten and water-soluble compounds released by gluten hydrolysis S. Ajaa, G. Pe´rezb, C.M. Rosella,* a
Department of Food Science, Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA), Apdo Correos 73, P.O. Box 73, 46100 Burjassot, Valencia, Spain b Facultad de Ciencias Agropecuarias, Universidad Nacional de Co´rdoba, C.C. 509, Co´rdoba 5000, Argentina Received 9 May 2003; revised 7 August 2003; accepted 6 October 2003
Abstract Wheat damage by heteropterous insects produces gluten hydrolysis giving different degradation products. Gluten content and gluten quality were assessed as a gluten index after incubation of the wet gluten for different intervals (0, 1, 2, 3, 7 and 24 h). Simultaneously, the water soluble products released by gluten hydrolysis during incubation were analysed by size-exclusion high performance liquid chromatography and SDS-PAGE. The results indicated that the amount of wet gluten remained constant even in the case of gluten isolated from damaged wheat, whereas the gluten index of damaged gluten showed a steady decrease with the incubation time suggesting an intense protein hydrolysis. A large amount of water soluble compounds were released from damaged gluten, increasing the relative proportion of compounds with molecular weights between 15,000 and 30,000 during the first 3 h of incubation. The SDS-PAGE studies under non-reducing conditions revealed presence of six new bands from Mr 42,000– 27,000 at 3 h of incubation and they showed a progressive increase in their intensity with incubation that progressively increased in intensity. The presence of some protein aggregates with Mr higher than 200,000 suggested the endoproteolytic activity of the insect proteases, and the analysis of the aggregates under reducing conditions indicated that they were linked by disulphide bonds. The gluten index is proposed as a parameter for objectively determining the insect attack. q 2003 Elsevier Ltd. All rights reserved. Keywords: Bug damaged wheat; Gluten; Gluten index; Water-soluble proteins; size-exclusion high performance liquid chromatography; SDS-PAGE
1. Introduction It is widely known that the preharvest attack of wheat by some Heteropterous insects yields grain with reduced breadmaking quality (Hariri et al., 2000; Lorenz and Meredith, 1988; Swallow and Every, 1991). This damage has been attributed to Nysius huttoni in New Zealand and to some species of Aelia and Eurygaster in Europe, Middle East and North Africa (Cressey et al., 1987; Every et al., 1992; Lorenz and Meredith, 1988). In Spain the most frequent species responsible for wheat damage are Aelia germari and Eurygaster austriaca (Infiesta et al., 1999). Abbreviations: IOD, integrated optical density; SE HPLC, size exclusion-high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis. * Corresponding author. Tel.: þ 34-96-390-0022; fax: þ34-96-363-6301. E-mail address: [email protected] (C.M. Rosell). 0733-5210/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2003.10.001
In feeding, the insect inserts its mouth parts into the immature grain and then sucks the milky juices (Every et al., 1990). The resulting mature wheat grains are partially empty in a large area below the puncture site, and in the surrounding that area the protein matrix is absent (Rosell et al., 2002a). Wheat flour from damaged wheat leads to sticky and weak doughs, and loaves of reduced volume and unacceptable texture (Hariri et al., 2000; Karababa and Nazmi Ozan, 1998; Matsoukas and Morrison, 1990). Nevertheless, the damaged wheat does not show abnormal hectolitre weight, thousand-kernel weight and protein content (Every et al., 1990; Lorenz and Meredith, 1988; Rosell et al., 2002a); even usual values of diastatic and alpha-amylase activity have been reported (Every et al., 1990; Rosell et al., 2002a). The unique characteristic of bugdamaged wheat is a disrupted protein structure (Cressey, 1987; Kretovich, 1944). It has been reported that insect infestation affects the glutenin and gliadin fraction of wheat proteins, with
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increased specificity towards the high molecular weight glutenin subunits (Cressey and McStay, 1987; Rosell et al., 2002b; Sivri et al., 1999). Reverse-phase high performance liquid chromatography (Rosell et al., 2002b; Sivri et al., 1999, 2002), free zone capillary electrophoresis (Aja et al., 2002; Rosell et al., 2002b), size-exclusion, high performance liquid chromatography (SE-HPLC) (Rosell et al., 2002b), and gel electrophoresis (PAGE and SDS-PAGE) (Sivri et al., 1998) have been applied to assess protein degradation due to insect attack. Little attention has been paid to the products released during protein hydrolysis. The degradation products from gluten hydrolysis are water or alcohol soluble proteins (Kretovich, 1944) in the case of Eurygaster and Aelia infestation, while incubation after Nysius attack does not lead to an increase in the amount of water soluble nitrogen (Swallow and Every, 1991). In fact, a quantitative test has been reported for Nysius proteinase in bug damaged wheat, which essentially measures the amount of aqueous SDS-soluble gluten protein after incubation of gluten with the enzyme extract from bug-damaged wheat (Every, 1991, 1992, 1993). However for Aelia and Eurygaster no further characterisation of the water soluble products has been reported, although it could be an important way to determine specific products for developing rapid detection assays. The aim of the present study was to analyse the water soluble products released on incubation of wet gluten from bug-damaged grain. In addition, the progressive changes in gluten quality were assessed using the gluten index.
2. Materials and methods Sound and damaged wheat grains from Bolero cultivar were provided by La Meta (Le´rida, Spain). The protein contents of the sound and visibly damaged wheats were 12.0 and 11% (based on 14% moisture content) for the sound wheat and visually damaged wheat, respectively. Chemical reagents of the highest purity were purchased from Sigma (St Louis, MO). 2.1. Gluten determination and proteolytic degradation assessment in wheat samples Wholemeal flour was prepared on a falling number mill type 3100. Wet gluten and gluten index were determined according to the AACC standard method (AACC, 1995). Washed gluten was kept in a shaking water bath at 37 8C for different time intervals, and subsequently wet gluten and the gluten index were determined by the standard methods. Proteolytic degradation was quantified by using a Chopin Alveograph (Tripette et Renaud, Paris, France) as previously described by Berger et al. (1974) and Rosell et al. (2002a,b) where a good correlation exists between deformation energy ðWÞ change after the dough is allowed to stand at 25 8C for 3 h and the proteolytic activity.
One unit of enzyme activity was arbitrarily defined as the reduction of the deformation energy after 3 h of incubation at 25 8C. No proteolytic activity was detected in the sound wheat, whereas the damaged wheat showed a proteolytic activity of 1.64 mU/g of wheat. 2.2. Extraction of the water-soluble fraction from incubated gluten Wet gluten from damaged wheat and sound wheat were obtained by using the Glutomatic (Perten, Huddinge, Sweden). Wet gluten (200 mg) was incubated at 37 8C in a shaking water bath for different time intervals; and then suspended in 1.0 ml of distilled water, and mixed for 5 mins on a vortex mixer and centrifuged at 15,700 £ g for 2 min. To ensure the absence of any remaining soluble starch the supernatant containing all the water-soluble compounds was mixed with three volumes of ethanol, kept overnight at 4 8C and then centrifuged at 15,700 £ g for 5 min. The supernatant was freeze-dried and stored for further analysis. 2.3. SE-HPLC analysis An Agilent 1100 Liquid Chromatograph was used for all the HPLC separations. Samples previously freeze dried were dissolved in 100 ml of distilled water. Size exclusion separation was performed by injecting 20 ml of sample at 0.4 ml/min of acetonitrile:water (20:80) containing 0.05% (w/v) trifluoroacetic acid into a TosoHaas TSK-gele G3000 PWXL column (TosoHaas GmbH, Stuttgart, Germany). Protein elution was monitored at 210 nm. Different molecular weight proteins were used for assessing the molecular weight of the different eluted fractions. The standard proteins used for calibrating the column were: apoferritin (443,000), beta-amylase (200,000), alcohol dehydrogenase (150,000), albumin (66,000), carbonic anhydrase (29,000), myoglobin (17,600), and cytochrome C (12,400). Quantification was performed using the chromatograph data analysis software (Hewlett Packard HPLC Chemstation ver. A.05). The chromatogram integration parameters were uniformly applied to all the chromatograms to quantify the distribution of the molecular weight of compounds in the water-soluble fraction released during gluten incubation. 2.4. Gel electrophoresis Protein composition of water extracts was analysed by SDS-PAGE (stacking gel of 4% (w/v) acrylamide and resolving gel of 12% (w/v) acrylamide) according to Laemmli (1970). A Mini Protean II Dual Slab Cell (BioRad Laboratories, Hercules, USA) was employed to perform electrophoretic runs working at constant voltage (150 V) until the front reached the end of the gel. Water extracts were dissolved in 0.125 M Tris/HCl (pH 6.8) containing 2% SDS, 10% glycerol, 0.05% bromophenol
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blue and 2% b-mercaptoethanol (reducing conditions) or without b-mercaptoethanol (non-reducing conditions). The protein bands were stained using silver stain.
using the Tukey test, and the significant differences were calculated at P , 0:05:
2.5. Densitometry and quantification of protein bands
3. Results and discussion
Gels were analysed by densitometry in an image master VDS (Pharmacia Biotech, Inc., Uppsala, Sweden) using the software image master VDS. A blank lane was used to obtain the background signal. The area of the protein band (integrated optical density, IOD) was represented by the expression
3.1. Effect of insect damage on the gluten characteristics
IOD ¼ ðmean intensity 2 backgroundÞ £ band area The standard curve for silver staining IOD vs quantity of protein was performed using carbonic anhydrase (C 7025 Sigma Chemical Co., St Louis, MO, USA) as standard. The silver stained IOD showed a linear response between 0.1 and 10 mg of protein with 6 a regression coefficient r 2 ¼ 0:915; and a lineal equation y ¼ 639:5 £ þ205:49: At least three determinations per point were made and average values were determined. 2.6. Statistical analysis All reported results are the means of at least four replicates. Results were analysed by the one-way analysis of variance procedure using Infostat, Statistical software (Facultad de Ciencias Agropecuarias, Universidad Nacional de Co´rdoba, Argentine). Means were compared
The gluten softening effect promoted by the insect infestation of wheat and a subjective method to quantify its effect has been reported (Cressey and McStay, 1987; Handford, 1967). In this study, although the objective was to analyse the water soluble products from protein hydrolysis, it seemed necessary to first determine the characteristics of the gluten proteins from sound and damaged wheat. This analysis was performed by assessing the wet gluten content and gluten quality during incubation at different time intervals as described in Section 2.1. Fig. 1 shows the effect of insect damage on the amount of wet gluten compared to that of sound wheat. No significant differences ðP , 0:05Þ were found in the amount of wet gluten as a consequence of insect damage. These results could be attributed to cultivar variability, since genetic factors and environmental conditions determine the wheat grain composition (Rosell et al., 2002a; Sivri et al., 2002). However when damaged kernels are carefully selected, abnormally low values of wet gluten are usually found in insect infested wheat (Karababa and Nazmi Ozan, 1998; Kretovich, 1944; Lorenz and Meredith, 1988). The amount of wet gluten remained constant during the incubation time, even in the case of the gluten isolated from damaged wheat. A slight decrease was observed after 24 h incubation in
Fig. 1. Effect of incubation at 37 8C on the amount of wet gluten (WG) and the quality of gluten (gluten index, GI) from damaged and sound wheat.
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the sound and damaged samples. These results indicate that the hydrolysis effect promoted by the insect attack is mainly of endoproteolytic type as previously stated by Cressey and McStay (1987), since the amount of wet gluten remains constant. A different pattern was observed for the gluten index. No significant differences were observed in the gluten index when fresh gluten from sound and damaged wheat was analysed. In the sound wheat the gluten index underwent a significant increase within the first 30 min of incubation, then remained constant with time decreasing only after 24 h incubation. The initial increase is indicative of polymerization of gluten proteins, that progressively increases and as a consequence increase the proportion of gluten retained on the sieve. Different wheat varieties have been tested and the same trend was always observed (results not shown). Thus gluten index values can only be attained from wet gluten rested at least 30 minutes after washing, otherwise a great variation within a sample will be obtained. With gluten isolated from damaged wheat, only a small increase in the gluten index was seen during the first 15 min incubation, but beyond that time a steady decrease was obtained, suggesting a very rapid hydrolysis process involving a size redistribution of the gluten proteins (Rosell et al., 2002b). Microbial counts on plate count agar after incubation at 30 8C for 24 h did not reveal any contamination; thus no microbial hydrolysis was occurring during the incubation. A comparison of the gluten index values at 30 min and 90 min could serve as a measure of protein degradation since sound wheat gives a constant gluten index during that
period in contrast to damaged wheat, which shows a reduced gluten index after resting. 3.2. Characterisation of the water soluble compounds by SE-HPLC Although no evident change was observed in the amount of wet gluten as a consequence of the insect damage, water soluble compounds were extracted and analysed by size exclusion chromatography. Similar SE-HPLC chromatogram patterns were observed for water soluble compounds isolated from damaged and undamaged gluten (Fig. 2). No statistically significant differences ðP , 0:05Þ were observed in the total area beneath the chromatograms, as was expected, since damaged wheat does not have abnormal values of protein content, thousand kernel weight, or specific weight (Karababa and Nazmi Ozan, 1998; Lorenz and Meredith, 1988; Rosell et al., 2002a), and no significant differences have been found in the alcohol soluble polymeric proteins and only a slightly lower gliadin content has been detected (Rosell et al., 2002b). However, a completely different chromatographic profile was observed after a 3-h incubation. Sound wheat showed only a slight increase in some peaks, which could be attributed to endogenous protease. Conversely, a great increase in the amount of water soluble compounds was observed in the damaged sample. Despite no variation being detected in the wet gluten, some of the products released during hydrolysis were soluble in water, and had a wide range of molecular weight. This result agrees with previous studies focused on the modification of gliadins and glutenins from
Fig. 2. Size exclusion HPLC separation of the water-soluble fraction isolated from damaged and sound wheat gluten. Overlaid chromatograms of unincubated (—) and 3 h incubated (· · ·) samples.
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Fig. 3. Effect of insect damage on the amount (in mAU s) of water-soluble compounds analysed by SE-HPLC. The water-soluble fractions were isolated from damaged (empty circles) and sound (filled circles) wheat gluten during incubation at 37 8C.
damaged wheat during incubation, showing a considerable decrease after incubation, the greatest degradation occuring with the high molecular weight glutenin subunits (Rosell et al., 2002b; Sivri et al., 1998; 2002). The sum of the total area beneath the chromatogram was used to quantify the total amount of water soluble compounds released as a consequence of the gluten hydrolysis (Fig. 3). The incubation of sound gluten barely produced an increase in the amount of water soluble protein compounds, only evident after 7 h of incubation. In contrast, the damaged gluten liberated a high amount of water soluble compounds even after a short incubation. When the size distribution of the water soluble compounds as analysed (Table 1), a modification of the distribution during incubation was observed, to an extent that was different in sound and damaged wheats. The majority of the water soluble products released from gluten
hydrolysis had a molecular size greater than 30,000 and a high proportion of molecular size smaller than 10,000 was also found. Small but statistically significant variations were observed in the size distribution of the water soluble compounds from sound gluten during incubation. No variation in the percentage of the fraction with molecular weight higher than 70,000 was detected during the incubation of sound and damaged gluten. Regarding damaged gluten, a progressive decrease in the relative proportion of the fractions with molecular weight between 70,000 and 30,000 and 15,000 – 10,000 was observed during incubation. In contrast, the percentage of the fractions with molecular weight between 30,000 and 20,000 and lower than 10,000 showed an increase during the same period. In the range 20,000 –15,000 the fraction from sound gluten showed a small increase at 30 min and no significant change was observed thereafter. In contrast the fractions in the same
Table 1 Molecular weight distribution of the water-soluble fraction from sound and damaged wheat gluten during incubation at 37 8C determined by SE-HPLC Molecular weight
.70,000 70,000–30,000 30,000–20,000 20,000–15,000 15,000–10,000 ,10,000
Sound wheat Incubation time (min)
Damaged wheat Incubation time (min)
0
30
60
120
180
0
30
60
120
180
61.0b 26.2b 1.2c 0.0e 3.8b 7.8c
61.1b 28.1a 1.2c 0.8c 3.2c 5.6f
59.7b 29.1a 1.4c 0.8c 2.9c,d 6.1e
61.1b 27.6a 1.7c,d 0.9c 2.2e 6.4e
59.6b 29.8a 0.8c,e 1.0c 2.6d 7.3d
63.3a 29.0a 0.0f 0.0e 4.7a 3.0g
65.5a 24.9c 1.0c 0.5d 1.6f 6.5e
65.1a 23.2d 2.2b 1.3b 1.8f 6.5e
63.7a 21.1e 2.4b 2.4a 1.8f 8.5b
63.8a 17.8f 3.9a 2.5a 1.4f,g 10.6a
Values are expressed as percentage of the total areas. Different letters within a row mean significant differences ðP # 0:05Þ:
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range from damaged gluten showed a steady increase during the incubation. 3.3. SDS-PAGE of the water soluble compounds When the water soluble extracts were analysed by SDS-PAGE in non-reducing conditions (Fig. 4) only a band of Mr 35,000 appeared when the sound gluten was incubated during 0, 3 and 7 h and their IOD did not change significantly (144 ^ 54, 113 ^ 68 and 139 ^ 47, respectively). However, after 24 h, numerous and intense protein bands were present, which correspond to the hydrolysis products resulting from the intrinsic proteolytic activity of the sound gluten. Those results are in agreement with the findings of Bleukx et al. (1997), describing the proteolytic activity associated with vital gluten. The water soluble compounds extracted from incubated damaged gluten showed six bands between Mr 42,000 and 27,000 that progressively increased in intensity during the incubation (1.80 ^ 0.13-fold between 3 and 7 h). In addition, two bands of Mr lower than 20,000 appeared but their intensity did not change after 7 h of incubation. Also a band retained between the stacking gel and the resolving gel
was observed at 3 and 7 h and most likely represented aggregated proteins. The intensity of this band slightly decreased under reducing conditions. Under these conditions number of bands appeared in the water soluble sample from damaged wheat, namely nine new bands of molecular weight between 45,000 and 20,000 indicating that large polymers composed of polypeptides linked by disulfide bonds were released. These results are in agreement with those observed when analysing wet gluten and indicate that the insect enzyme is an endoprotease like the Nysius proteinase reported by Cressey and McStay (1987) and Every (1993). Kretovitch (1994) provided the initial information relating to water soluble compounds from damaged wheat coming from the increase in water soluble nitrogen values. He also suggested that the compounds released could be peptones, peptides and amino acids. Our results show that proteins and peptide aggregates of high molecular weight (higher than 200,000) were also released during incubation and that they are water soluble.
4. Conclusion The measurement of the gluten index before and after resting could be a very useful way to detect damaged wheat produced by heteropterous insects. The hydrolysis of wheat proteins leads to the release of water soluble compounds and their amount increased with the incubation time. The proteins and peptides released have a wide range of molecular weight, from peptide aggregates linked by disulfide bonds higher than 200,000 to small peptides lower than 10,000.
Acknowledgements This study was financially supported by the European Union and Comisio´n Interministerial de Ciencia y Tecnologia Project (MCYT, AGL2001-1273; AGL2002-04093C03-02 ALI) and Consejo Superior de Investigaciones Cientificas (CSIC, Spain). G Pe´rez gratefully acknowledges the Agencia Espan˜ola de Cooperacio´n (AECI) for her grant. Authors would like to thank Carles Miralbes for providing wheat samples.
References
Fig. 4. Pattern of SDS-PAGE gels (12% acrylamide) under non-reducing conditions (upper gel) and reducing conditions (lower gel) of water-soluble fractions from damaged and sound wheat gluten. Lane 1: molecular mass marker Myosin 200,000; Phosphorylase b 97,400; Serum albumin 66,200; Ovalbumin 45,000; Carbonic anhydrase 31,000; Soybean trypsin inhibitor 21,500. Lane 2–5: water soluble fractions from sound wheat incubated 0, 3, 7 and 24 h. Lane 6– 9: water soluble fractions from damaged wheat incubated 0, 3, 7 and 24 h.
AACC, 1995. Approved Methods, American Association of Cereal Chemists, St Paul, MN, Standard no. 46-12, pp. 56B–81B. Aja, S., Rosell, C.M., Benedito, C., 2002. Detection of cereal protein alterations by using free zone capillary electrophoresis, Proceedings of ICC Conference Novel Raw Materials, Technologies and ProductsNew Challenge for the Quality Control, Budapest, pp. 237 –244. Berger, M., Grandvoinnet, P., de la Gue´rivie`re, J.F., Bussie`re, G., 1974. Tests de rhe´ologie pratique utilisables pour l’appre´ciation des activite´s prote´olitiques. Ann. Technol. Agric. 23, 233–239.
S. Aja et al. / Journal of Cereal Science 39 (2004) 187–193 Bleukx, W., Roels, S.P., Delcour, J.A., 1997. On the presence and activities of proteolytic enzymes in vital wheat gluten. J. Cereal Sci. 26, 183–193. Cressey, P.J., 1987. Wheat bug damage in New Zealand wheats. Some properties of a glutenin hydrolysing enzyme in bug-damage wheat. J. Sci. Food Agric. 41, 159 –165. Cressey, P.J., McStay, C.L., 1987. Wheat bug damage in New Zealand wheats. Development of a simple SDS sedimentation test for bug damage. J. Sci. Food Agric. 38, 357 –366. Cressey, P.J., Farrell, J.A.K., Stufkens, M.W., 1987. Identification of an insect species causing bug damage in New Zealand wheats. New Zealand J. Agric. Res. 30, 209 –212. Every, D., 1991. General proteinase assay by formation of SDS-protein gels of proteolysed substrate proteins. Anal. Biochem. 197, 208 –212. Every, D., 1992. Relationship of bread baking quality to levels of visible wheat-bug damage and insect proteinase activity in wheat. J. Cereal Sci. 16, 183 –193. Every, D., 1993. Purification and characterization of a glutenin hydrolysing proteinase from wheat damaged by New Zealand wheat bug, Nysius huttoni. J. Cereal Sci. 18, 239 –250. Every, D., Farrell, J.A.K., Stufkens, M.W., 1990. Wheat-bug damage in New Zealand wheats: the feeding mechanism of Nysius huttoni and its effect on the morphological and physiological development of wheat. J. Sci. Food Agric. 50, 297 –300. Every, D., Farrell, J.A., Stufkens, M.W., 1992. Bug damage in New Zealand wheat grain: the role of various hetetopterous insects. New Zealand J. Crop Hortic. Sci. 20, 305–312. Hanford, J., 1967. The proteolytic enzymes of wheat and flour and their effect on bread quality in the United Kingdom. Cereal Chem. 44, 499–511. Hariri, G., Williams, P.C., El-Haramein, F.J., 2000. Influence of pentatomid insect on the physical dough properties and two-layered flat bread baking quality of Syrian wheat. J. Cereal Sci. 31, 111–118.
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Infiesta, E., Gouda, G., Monleon, T., Valero, J., Gordun, E., 1999. Aelia y Eurygaster en muestras de trigos espanoles de 1997. Molineria y Panaderia. 3, 61–66. Karababa, E., Nazmi Ozan, A., 1998. Effect of wheat bug (Eurygaster integriceps) damage on quality of a wheat variety grown in Turkey. J. Sci. Food Agric. 77, 399 –403. Kretovich, V.L., 1944. Biochemistry of the damage to grain by wheat bug. Cereal Chem. 21, 1–16. Laemmli, V., 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227, 681 –684. Lorenz, K., Meredith, P., 1988. Insect damaged wheat: history of the problem, effects on baking quality, remedies. Lebensm Wiss und Technol. 21, 183 –187. Matsoukas, N.P., Morrison, W., 1990. Breadmaking quality of ten Greek breadwheats baking and storage tests on bread made by long fermentation and activated (chemical) dough development process, and the effects of bug-damaged wheat. J. Sci. Food Agric. 53, 363–377. Rosell, C.M., Aja, S., Sadowska, J., 2002a. Amylase activities in insect (Aelia and Eurygaster) damaged wheat. J. Sci. Food Agric. 9, 977–982. Rosell, C.M., Aja, S., Bean, S., Lookhart, G., 2002b. Effect of Aelia and Eurygaster damage on wheat proteins. Cereal Chem. 79, 801–805. Sivri, D., Koksel, H., Bushuk, W., 1998. Effect of wheat bug (Eurygaster maura) proteolytic enzymes on electrophoretic properties of gluten proteins. New Zealand J. Crop Hortic. Sci. 26, 117 –125. Sivri, D., Sapirstein, H.D., Koksel, H., Bushuk, W., 1999. Effects of wheat bug (Eurygaster maura) protease on glutenin proteins. Cereal Chem. 76, 816–820. Sivri, D., Sapirstein, H.D., Bushuk, W., Koksel, H., 2002. Wheat intercultivar differences in susceptibility of glutenin protein to effects of bug (Eurygaster integriceps) protease. Cereal Chem. 79, 41 –44. Swallow, W.H., Every, D., 1991. Insect enzyme damage to wheat. Cereal Foods World 36, 505–508.
Wheat damage by Aelia spp. and Erygaster spp.: effects on gluten and water-soluble compounds released by gluten hydrolysis S. Ajaa, G. Pe´rezb, C.M. Rosella,* a
Department of Food Science, Instituto de Agroquı´mica y Tecnologı´a de Alimentos (IATA), Apdo Correos 73, P.O. Box 73, 46100 Burjassot, Valencia, Spain b Facultad de Ciencias Agropecuarias, Universidad Nacional de Co´rdoba, C.C. 509, Co´rdoba 5000, Argentina Received 9 May 2003; revised 7 August 2003; accepted 6 October 2003
Abstract Wheat damage by heteropterous insects produces gluten hydrolysis giving different degradation products. Gluten content and gluten quality were assessed as a gluten index after incubation of the wet gluten for different intervals (0, 1, 2, 3, 7 and 24 h). Simultaneously, the water soluble products released by gluten hydrolysis during incubation were analysed by size-exclusion high performance liquid chromatography and SDS-PAGE. The results indicated that the amount of wet gluten remained constant even in the case of gluten isolated from damaged wheat, whereas the gluten index of damaged gluten showed a steady decrease with the incubation time suggesting an intense protein hydrolysis. A large amount of water soluble compounds were released from damaged gluten, increasing the relative proportion of compounds with molecular weights between 15,000 and 30,000 during the first 3 h of incubation. The SDS-PAGE studies under non-reducing conditions revealed presence of six new bands from Mr 42,000– 27,000 at 3 h of incubation and they showed a progressive increase in their intensity with incubation that progressively increased in intensity. The presence of some protein aggregates with Mr higher than 200,000 suggested the endoproteolytic activity of the insect proteases, and the analysis of the aggregates under reducing conditions indicated that they were linked by disulphide bonds. The gluten index is proposed as a parameter for objectively determining the insect attack. q 2003 Elsevier Ltd. All rights reserved. Keywords: Bug damaged wheat; Gluten; Gluten index; Water-soluble proteins; size-exclusion high performance liquid chromatography; SDS-PAGE
1. Introduction It is widely known that the preharvest attack of wheat by some Heteropterous insects yields grain with reduced breadmaking quality (Hariri et al., 2000; Lorenz and Meredith, 1988; Swallow and Every, 1991). This damage has been attributed to Nysius huttoni in New Zealand and to some species of Aelia and Eurygaster in Europe, Middle East and North Africa (Cressey et al., 1987; Every et al., 1992; Lorenz and Meredith, 1988). In Spain the most frequent species responsible for wheat damage are Aelia germari and Eurygaster austriaca (Infiesta et al., 1999). Abbreviations: IOD, integrated optical density; SE HPLC, size exclusion-high performance liquid chromatography; SDS-PAGE, sodium dodecyl sulphate-polyacrylamide gel electrophoresis. * Corresponding author. Tel.: þ 34-96-390-0022; fax: þ34-96-363-6301. E-mail address: [email protected] (C.M. Rosell). 0733-5210/$ - see front matter q 2003 Elsevier Ltd. All rights reserved. doi:10.1016/j.jcs.2003.10.001
In feeding, the insect inserts its mouth parts into the immature grain and then sucks the milky juices (Every et al., 1990). The resulting mature wheat grains are partially empty in a large area below the puncture site, and in the surrounding that area the protein matrix is absent (Rosell et al., 2002a). Wheat flour from damaged wheat leads to sticky and weak doughs, and loaves of reduced volume and unacceptable texture (Hariri et al., 2000; Karababa and Nazmi Ozan, 1998; Matsoukas and Morrison, 1990). Nevertheless, the damaged wheat does not show abnormal hectolitre weight, thousand-kernel weight and protein content (Every et al., 1990; Lorenz and Meredith, 1988; Rosell et al., 2002a); even usual values of diastatic and alpha-amylase activity have been reported (Every et al., 1990; Rosell et al., 2002a). The unique characteristic of bugdamaged wheat is a disrupted protein structure (Cressey, 1987; Kretovich, 1944). It has been reported that insect infestation affects the glutenin and gliadin fraction of wheat proteins, with
188
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increased specificity towards the high molecular weight glutenin subunits (Cressey and McStay, 1987; Rosell et al., 2002b; Sivri et al., 1999). Reverse-phase high performance liquid chromatography (Rosell et al., 2002b; Sivri et al., 1999, 2002), free zone capillary electrophoresis (Aja et al., 2002; Rosell et al., 2002b), size-exclusion, high performance liquid chromatography (SE-HPLC) (Rosell et al., 2002b), and gel electrophoresis (PAGE and SDS-PAGE) (Sivri et al., 1998) have been applied to assess protein degradation due to insect attack. Little attention has been paid to the products released during protein hydrolysis. The degradation products from gluten hydrolysis are water or alcohol soluble proteins (Kretovich, 1944) in the case of Eurygaster and Aelia infestation, while incubation after Nysius attack does not lead to an increase in the amount of water soluble nitrogen (Swallow and Every, 1991). In fact, a quantitative test has been reported for Nysius proteinase in bug damaged wheat, which essentially measures the amount of aqueous SDS-soluble gluten protein after incubation of gluten with the enzyme extract from bug-damaged wheat (Every, 1991, 1992, 1993). However for Aelia and Eurygaster no further characterisation of the water soluble products has been reported, although it could be an important way to determine specific products for developing rapid detection assays. The aim of the present study was to analyse the water soluble products released on incubation of wet gluten from bug-damaged grain. In addition, the progressive changes in gluten quality were assessed using the gluten index.
2. Materials and methods Sound and damaged wheat grains from Bolero cultivar were provided by La Meta (Le´rida, Spain). The protein contents of the sound and visibly damaged wheats were 12.0 and 11% (based on 14% moisture content) for the sound wheat and visually damaged wheat, respectively. Chemical reagents of the highest purity were purchased from Sigma (St Louis, MO). 2.1. Gluten determination and proteolytic degradation assessment in wheat samples Wholemeal flour was prepared on a falling number mill type 3100. Wet gluten and gluten index were determined according to the AACC standard method (AACC, 1995). Washed gluten was kept in a shaking water bath at 37 8C for different time intervals, and subsequently wet gluten and the gluten index were determined by the standard methods. Proteolytic degradation was quantified by using a Chopin Alveograph (Tripette et Renaud, Paris, France) as previously described by Berger et al. (1974) and Rosell et al. (2002a,b) where a good correlation exists between deformation energy ðWÞ change after the dough is allowed to stand at 25 8C for 3 h and the proteolytic activity.
One unit of enzyme activity was arbitrarily defined as the reduction of the deformation energy after 3 h of incubation at 25 8C. No proteolytic activity was detected in the sound wheat, whereas the damaged wheat showed a proteolytic activity of 1.64 mU/g of wheat. 2.2. Extraction of the water-soluble fraction from incubated gluten Wet gluten from damaged wheat and sound wheat were obtained by using the Glutomatic (Perten, Huddinge, Sweden). Wet gluten (200 mg) was incubated at 37 8C in a shaking water bath for different time intervals; and then suspended in 1.0 ml of distilled water, and mixed for 5 mins on a vortex mixer and centrifuged at 15,700 £ g for 2 min. To ensure the absence of any remaining soluble starch the supernatant containing all the water-soluble compounds was mixed with three volumes of ethanol, kept overnight at 4 8C and then centrifuged at 15,700 £ g for 5 min. The supernatant was freeze-dried and stored for further analysis. 2.3. SE-HPLC analysis An Agilent 1100 Liquid Chromatograph was used for all the HPLC separations. Samples previously freeze dried were dissolved in 100 ml of distilled water. Size exclusion separation was performed by injecting 20 ml of sample at 0.4 ml/min of acetonitrile:water (20:80) containing 0.05% (w/v) trifluoroacetic acid into a TosoHaas TSK-gele G3000 PWXL column (TosoHaas GmbH, Stuttgart, Germany). Protein elution was monitored at 210 nm. Different molecular weight proteins were used for assessing the molecular weight of the different eluted fractions. The standard proteins used for calibrating the column were: apoferritin (443,000), beta-amylase (200,000), alcohol dehydrogenase (150,000), albumin (66,000), carbonic anhydrase (29,000), myoglobin (17,600), and cytochrome C (12,400). Quantification was performed using the chromatograph data analysis software (Hewlett Packard HPLC Chemstation ver. A.05). The chromatogram integration parameters were uniformly applied to all the chromatograms to quantify the distribution of the molecular weight of compounds in the water-soluble fraction released during gluten incubation. 2.4. Gel electrophoresis Protein composition of water extracts was analysed by SDS-PAGE (stacking gel of 4% (w/v) acrylamide and resolving gel of 12% (w/v) acrylamide) according to Laemmli (1970). A Mini Protean II Dual Slab Cell (BioRad Laboratories, Hercules, USA) was employed to perform electrophoretic runs working at constant voltage (150 V) until the front reached the end of the gel. Water extracts were dissolved in 0.125 M Tris/HCl (pH 6.8) containing 2% SDS, 10% glycerol, 0.05% bromophenol
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blue and 2% b-mercaptoethanol (reducing conditions) or without b-mercaptoethanol (non-reducing conditions). The protein bands were stained using silver stain.
using the Tukey test, and the significant differences were calculated at P , 0:05:
2.5. Densitometry and quantification of protein bands
3. Results and discussion
Gels were analysed by densitometry in an image master VDS (Pharmacia Biotech, Inc., Uppsala, Sweden) using the software image master VDS. A blank lane was used to obtain the background signal. The area of the protein band (integrated optical density, IOD) was represented by the expression
3.1. Effect of insect damage on the gluten characteristics
IOD ¼ ðmean intensity 2 backgroundÞ £ band area The standard curve for silver staining IOD vs quantity of protein was performed using carbonic anhydrase (C 7025 Sigma Chemical Co., St Louis, MO, USA) as standard. The silver stained IOD showed a linear response between 0.1 and 10 mg of protein with 6 a regression coefficient r 2 ¼ 0:915; and a lineal equation y ¼ 639:5 £ þ205:49: At least three determinations per point were made and average values were determined. 2.6. Statistical analysis All reported results are the means of at least four replicates. Results were analysed by the one-way analysis of variance procedure using Infostat, Statistical software (Facultad de Ciencias Agropecuarias, Universidad Nacional de Co´rdoba, Argentine). Means were compared
The gluten softening effect promoted by the insect infestation of wheat and a subjective method to quantify its effect has been reported (Cressey and McStay, 1987; Handford, 1967). In this study, although the objective was to analyse the water soluble products from protein hydrolysis, it seemed necessary to first determine the characteristics of the gluten proteins from sound and damaged wheat. This analysis was performed by assessing the wet gluten content and gluten quality during incubation at different time intervals as described in Section 2.1. Fig. 1 shows the effect of insect damage on the amount of wet gluten compared to that of sound wheat. No significant differences ðP , 0:05Þ were found in the amount of wet gluten as a consequence of insect damage. These results could be attributed to cultivar variability, since genetic factors and environmental conditions determine the wheat grain composition (Rosell et al., 2002a; Sivri et al., 2002). However when damaged kernels are carefully selected, abnormally low values of wet gluten are usually found in insect infested wheat (Karababa and Nazmi Ozan, 1998; Kretovich, 1944; Lorenz and Meredith, 1988). The amount of wet gluten remained constant during the incubation time, even in the case of the gluten isolated from damaged wheat. A slight decrease was observed after 24 h incubation in
Fig. 1. Effect of incubation at 37 8C on the amount of wet gluten (WG) and the quality of gluten (gluten index, GI) from damaged and sound wheat.
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the sound and damaged samples. These results indicate that the hydrolysis effect promoted by the insect attack is mainly of endoproteolytic type as previously stated by Cressey and McStay (1987), since the amount of wet gluten remains constant. A different pattern was observed for the gluten index. No significant differences were observed in the gluten index when fresh gluten from sound and damaged wheat was analysed. In the sound wheat the gluten index underwent a significant increase within the first 30 min of incubation, then remained constant with time decreasing only after 24 h incubation. The initial increase is indicative of polymerization of gluten proteins, that progressively increases and as a consequence increase the proportion of gluten retained on the sieve. Different wheat varieties have been tested and the same trend was always observed (results not shown). Thus gluten index values can only be attained from wet gluten rested at least 30 minutes after washing, otherwise a great variation within a sample will be obtained. With gluten isolated from damaged wheat, only a small increase in the gluten index was seen during the first 15 min incubation, but beyond that time a steady decrease was obtained, suggesting a very rapid hydrolysis process involving a size redistribution of the gluten proteins (Rosell et al., 2002b). Microbial counts on plate count agar after incubation at 30 8C for 24 h did not reveal any contamination; thus no microbial hydrolysis was occurring during the incubation. A comparison of the gluten index values at 30 min and 90 min could serve as a measure of protein degradation since sound wheat gives a constant gluten index during that
period in contrast to damaged wheat, which shows a reduced gluten index after resting. 3.2. Characterisation of the water soluble compounds by SE-HPLC Although no evident change was observed in the amount of wet gluten as a consequence of the insect damage, water soluble compounds were extracted and analysed by size exclusion chromatography. Similar SE-HPLC chromatogram patterns were observed for water soluble compounds isolated from damaged and undamaged gluten (Fig. 2). No statistically significant differences ðP , 0:05Þ were observed in the total area beneath the chromatograms, as was expected, since damaged wheat does not have abnormal values of protein content, thousand kernel weight, or specific weight (Karababa and Nazmi Ozan, 1998; Lorenz and Meredith, 1988; Rosell et al., 2002a), and no significant differences have been found in the alcohol soluble polymeric proteins and only a slightly lower gliadin content has been detected (Rosell et al., 2002b). However, a completely different chromatographic profile was observed after a 3-h incubation. Sound wheat showed only a slight increase in some peaks, which could be attributed to endogenous protease. Conversely, a great increase in the amount of water soluble compounds was observed in the damaged sample. Despite no variation being detected in the wet gluten, some of the products released during hydrolysis were soluble in water, and had a wide range of molecular weight. This result agrees with previous studies focused on the modification of gliadins and glutenins from
Fig. 2. Size exclusion HPLC separation of the water-soluble fraction isolated from damaged and sound wheat gluten. Overlaid chromatograms of unincubated (—) and 3 h incubated (· · ·) samples.
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Fig. 3. Effect of insect damage on the amount (in mAU s) of water-soluble compounds analysed by SE-HPLC. The water-soluble fractions were isolated from damaged (empty circles) and sound (filled circles) wheat gluten during incubation at 37 8C.
damaged wheat during incubation, showing a considerable decrease after incubation, the greatest degradation occuring with the high molecular weight glutenin subunits (Rosell et al., 2002b; Sivri et al., 1998; 2002). The sum of the total area beneath the chromatogram was used to quantify the total amount of water soluble compounds released as a consequence of the gluten hydrolysis (Fig. 3). The incubation of sound gluten barely produced an increase in the amount of water soluble protein compounds, only evident after 7 h of incubation. In contrast, the damaged gluten liberated a high amount of water soluble compounds even after a short incubation. When the size distribution of the water soluble compounds as analysed (Table 1), a modification of the distribution during incubation was observed, to an extent that was different in sound and damaged wheats. The majority of the water soluble products released from gluten
hydrolysis had a molecular size greater than 30,000 and a high proportion of molecular size smaller than 10,000 was also found. Small but statistically significant variations were observed in the size distribution of the water soluble compounds from sound gluten during incubation. No variation in the percentage of the fraction with molecular weight higher than 70,000 was detected during the incubation of sound and damaged gluten. Regarding damaged gluten, a progressive decrease in the relative proportion of the fractions with molecular weight between 70,000 and 30,000 and 15,000 – 10,000 was observed during incubation. In contrast, the percentage of the fractions with molecular weight between 30,000 and 20,000 and lower than 10,000 showed an increase during the same period. In the range 20,000 –15,000 the fraction from sound gluten showed a small increase at 30 min and no significant change was observed thereafter. In contrast the fractions in the same
Table 1 Molecular weight distribution of the water-soluble fraction from sound and damaged wheat gluten during incubation at 37 8C determined by SE-HPLC Molecular weight
.70,000 70,000–30,000 30,000–20,000 20,000–15,000 15,000–10,000 ,10,000
Sound wheat Incubation time (min)
Damaged wheat Incubation time (min)
0
30
60
120
180
0
30
60
120
180
61.0b 26.2b 1.2c 0.0e 3.8b 7.8c
61.1b 28.1a 1.2c 0.8c 3.2c 5.6f
59.7b 29.1a 1.4c 0.8c 2.9c,d 6.1e
61.1b 27.6a 1.7c,d 0.9c 2.2e 6.4e
59.6b 29.8a 0.8c,e 1.0c 2.6d 7.3d
63.3a 29.0a 0.0f 0.0e 4.7a 3.0g
65.5a 24.9c 1.0c 0.5d 1.6f 6.5e
65.1a 23.2d 2.2b 1.3b 1.8f 6.5e
63.7a 21.1e 2.4b 2.4a 1.8f 8.5b
63.8a 17.8f 3.9a 2.5a 1.4f,g 10.6a
Values are expressed as percentage of the total areas. Different letters within a row mean significant differences ðP # 0:05Þ:
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range from damaged gluten showed a steady increase during the incubation. 3.3. SDS-PAGE of the water soluble compounds When the water soluble extracts were analysed by SDS-PAGE in non-reducing conditions (Fig. 4) only a band of Mr 35,000 appeared when the sound gluten was incubated during 0, 3 and 7 h and their IOD did not change significantly (144 ^ 54, 113 ^ 68 and 139 ^ 47, respectively). However, after 24 h, numerous and intense protein bands were present, which correspond to the hydrolysis products resulting from the intrinsic proteolytic activity of the sound gluten. Those results are in agreement with the findings of Bleukx et al. (1997), describing the proteolytic activity associated with vital gluten. The water soluble compounds extracted from incubated damaged gluten showed six bands between Mr 42,000 and 27,000 that progressively increased in intensity during the incubation (1.80 ^ 0.13-fold between 3 and 7 h). In addition, two bands of Mr lower than 20,000 appeared but their intensity did not change after 7 h of incubation. Also a band retained between the stacking gel and the resolving gel
was observed at 3 and 7 h and most likely represented aggregated proteins. The intensity of this band slightly decreased under reducing conditions. Under these conditions number of bands appeared in the water soluble sample from damaged wheat, namely nine new bands of molecular weight between 45,000 and 20,000 indicating that large polymers composed of polypeptides linked by disulfide bonds were released. These results are in agreement with those observed when analysing wet gluten and indicate that the insect enzyme is an endoprotease like the Nysius proteinase reported by Cressey and McStay (1987) and Every (1993). Kretovitch (1994) provided the initial information relating to water soluble compounds from damaged wheat coming from the increase in water soluble nitrogen values. He also suggested that the compounds released could be peptones, peptides and amino acids. Our results show that proteins and peptide aggregates of high molecular weight (higher than 200,000) were also released during incubation and that they are water soluble.
4. Conclusion The measurement of the gluten index before and after resting could be a very useful way to detect damaged wheat produced by heteropterous insects. The hydrolysis of wheat proteins leads to the release of water soluble compounds and their amount increased with the incubation time. The proteins and peptides released have a wide range of molecular weight, from peptide aggregates linked by disulfide bonds higher than 200,000 to small peptides lower than 10,000.
Acknowledgements This study was financially supported by the European Union and Comisio´n Interministerial de Ciencia y Tecnologia Project (MCYT, AGL2001-1273; AGL2002-04093C03-02 ALI) and Consejo Superior de Investigaciones Cientificas (CSIC, Spain). G Pe´rez gratefully acknowledges the Agencia Espan˜ola de Cooperacio´n (AECI) for her grant. Authors would like to thank Carles Miralbes for providing wheat samples.
References
Fig. 4. Pattern of SDS-PAGE gels (12% acrylamide) under non-reducing conditions (upper gel) and reducing conditions (lower gel) of water-soluble fractions from damaged and sound wheat gluten. Lane 1: molecular mass marker Myosin 200,000; Phosphorylase b 97,400; Serum albumin 66,200; Ovalbumin 45,000; Carbonic anhydrase 31,000; Soybean trypsin inhibitor 21,500. Lane 2–5: water soluble fractions from sound wheat incubated 0, 3, 7 and 24 h. Lane 6– 9: water soluble fractions from damaged wheat incubated 0, 3, 7 and 24 h.
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